The human calcitonin family peptide hormones calcitonin (CT), amylin (Amy), calcitonin-gene related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) exhibit diverse actions including regulation of bone remodeling (CT), vasodilation and cardioprotection (CGRP, AM, AM2/IMD), and regulation of blood glucose and food intake (Amy) by activating the cell surface class B G protein-coupled receptors (GPCRs) calcitonin receptor (CTR) and calcitonin-like receptor (CLR). Three receptor activity-modifying proteins (RAMP1-3) heterodimerize with CLR and CTR and determine their peptide preferences. The seven resulting receptors (six heterodimers and CTR alone) are proven or promising drug targets for diseases that are a significant burden on human health including migraine headache (CGRP receptor; CLR:RAMP1), diabetes and obesity (Amy receptors; CTR:RAMP1/2/3), osteoporosis (CT receptor; CTR), and cardiovascular disorders (AM receptors; CLR:RAMP2/3). These complex receptors are a paradigm for modulation of GPCR pharmacology by accessory membrane proteins, but our understanding of their selective peptide recognition mechanisms and distinct biological functions is incomplete. Our structural studies have highlighted the critical role of the receptor extracellular domains (ECDs) for peptide selectivity and provided important insights into how RAMP1 and RAMP2 alter CLR selectivity for CGRP and AM. Herein we continue to investigate the mechanisms by which RAMPs modulate CLR and CTR. We will test the hypothesis that allosteric modulation of CLR conformation by RAMPs is important for selectivity and based on our discovery that CTR N- glycosylation enhances its peptide affinity 10-fold we will test the hypothesis that this effect is also allosteric in nature. We will fill critical gaps in our knowledge by providing crystal structures of AM2/IMD, CT, and Amy bound to their receptor ECD complexes. We use biochemical, pharmacological, and X-ray crystallographic approaches for each of the specific aims. In Aim 1 we will probe the role of allostery in RAMP-altered CLR peptide selectivity through the use of novel altered selectivity antagonist CGRP and AM variants developed through rational design and screening of synthetic peptide combinatorial libraries. In Aim 2 we will determine how AM2/IMD binds CLR:RAMP1 and CLR:RAMP3 ECD complexes and how RAMP3 modulates CLR. In Aim 3 we will define how RAMPs and N-glycosylation of CTR ECD modulate CT and Amy binding. Successful completion of this project will lead to the following outcomes: 1) a better understanding of how each of the CT family peptides bind their receptors, 2) delineation of the role of allostery in RAMP function, 3) elucidation of the mechanisms by which N-glycans and RAMPs modulate CTR peptide binding, and 4) development of novel peptide antagonists with enhanced affinities and altered selectivities that may be of value as pharmacological tools for interrogating receptor biology and may inform drug development targeting the receptors.